1. Field of the Invention
This invention relates to a transformation system that includes a gene transfer vector containing a modified piggybac transposon (pB) and having the insertion of a marker construct containing an enhanced green fluorescent protein gene (EGFP) linked to a polyubiquitin promoter gene and a nuclear localizing sequence. The invention further relates to a helper vector containing a heat shock protein gene and to methods for using this system to transform eukaryotic cells as well as transgenic organisms produced using the system, especially insect cells and insects, respectively.
2. Description of the Related Art
The piggyBac transposable element from the cabbage looper moth, Trichoplusia ni (Cary et al., Virology, Volume 161, 8-17, 1989) has been shown to be an effective gene-transfer vector in the Mediterranean fruit fly, Ceratitis capitata (Handler et al., Proc. Natl. Acad. Sci. USA, Volume 95, 7520-7525, 1998). Use of an unmodified transposase helper under piggyBac promoter regulation indicates that piggyBac retains autonomous function in the medfly, since transcriptional regulation was maintained, as well as enzymatic activity. This observation was unique since all other successful insect germline transformations had been limited to dipteran species using vectors isolated from the same or another dipteran. The initial transformation of medfly (Loukeris et al., Science, Volume 270, 2002-2005, 1995) used the Minos vector from Drosophila hydei (Franz & Savakis, Nucl. Acids Res., Volume 19, 6646, 1991), and Aedes aegypti has been transformed from Hermes (Jasinskiene et al., Proc. Natl. Acad. Sci. USA, Volume 95, 3743-3747, 1998) from Musca domestica (Warren et al., Genet. Res. Camb., Volume 64, 87-97, 1994) and mariner (Coates et al., Proc. Natl. Acad. Sci. USA, Volume 95, 3748-3751, 1998) from Drosophila mauritiana (Jacobson et al., Proc. Natl. Acad. Sci. USA, Volume 83, 8684-8688, 1986). Drosophila melanogaster has been transformed as well by Hermes (O'Brochta et al., Insect Biochem. Molec. Biol., Volume 26, 739-753, 1996) mariner (Lidholm et al., Genetics, Volume 134, 859-868, 1993); Minos (Franz et al., Proc. Natl. Acad. Sci. USA, Volume 91, 4746-4750, 1994) and by the P and hobo transposons originally discovered in its own genome (Rubin and Spradling, 1989; Blackman et al., EMBO J., Volume 8, 211-217, 1989). Drosophila virilis also has been transformed by hobo (Lozovskaya et al., Genetics, Volume 143, 365-374, 1995; Gomez & Handler, Insect Mol. Biol., Volume 6, 1-8, 1997) and mariner (Lohe et al., Genetics, Volume 143, 365-374, 1996). While the restriction to dipteran vectors is due in part to the limited number of transposon systems available from non-dipteran species, phylogenetic limitations on transposon function is not unexpected considering the deleterious effects functional transposons may have on a host genome. This is, indeed, reflected by the high level of regulation placed on transposon movement among species, among strains within a host species, and even among cell types within an organism (Berg & Howe, Mobile DNA, American Society for Microbiology, Washington, D.C. 1989).
The ability of piggyBac to function in several dipteran species will be supportive of its use in a wider range of insects, if not other organisms. Most other vector systems function optimally, or have been only tested with their helper transposase under hsp70 promoter regulation. The transposition efficiency of most vectors has been also found to be influenced by the amount of internal DNA inserted, the position of this DNA within the vector, and the amount of subterminal DNA remaining in the vectors.
The widespread use of piggyBac will be limited by the availability of easily detectable and unambiguous transformant markers. Most Drosophila transformations, as well as the few nondrosophilid transformations reported have depended on transformant selection by rescue of a mutant visible phenotype, usually eye pigmentation (Ashburner et al., Insect Mol. Biol., Volume 7, 201-213, 1998). Unfortunately, most insect species have neither visible mutant strains, nor the cloned DNA for the wild type allele of the mutation, and these species require use of new dominant-acting marker genes that confer, preferably, a visible phenotype.
The present invention, discussed below, provides a system that includes vectors for transforming eukaryotic cells, derived from piggyBac transposons that are different from related art vectors. Furthermore, the present invention increases the transformation frequency by about eight-fold compared to other piggyBac transformation systems.